Integrative taxonomic revision of the genera Nesticella and Howaia in Japan with the description of five new species (Araneae, Nesticidae, Nesticellini) Author Ballarin, Francesco https://orcid.org/0000-0003-1417-2519 Systematic Zoology Laboratory, Department of Biological Sciences, Tokyo Metropolitan University, 1 - 1 Minami-Osawa, Hachioji-shi, 192 - 0397, Tokyo, Japan & Department of Zoology, Museo di Storia Naturale of Verona, Lungadige Porta Vittoria, 9, I- 37129 Verona, Italy ballarin.francesco@gmail.com Author Eguchi, Katsuyuki https://orcid.org/0000-0002-1054-1295 Systematic Zoology Laboratory, Department of Biological Sciences, Tokyo Metropolitan University, 1 - 1 Minami-Osawa, Hachioji-shi, 192 - 0397, Tokyo, Japan & Department of International Health and Medical Anthropology, Institute of Tropical Medicine, Nagasaki University, 1 - 12 - 4 Sakamoto, Nagasaki-shi, 852 - 8523, Nagasaki, Japan text ZooKeys 2023 2023-08-11 1174 219 272 http://dx.doi.org/10.3897/zookeys.1174.101251 journal article http://dx.doi.org/10.3897/zookeys.1174.101251 1313-2970-1174-219 608FAD80206A428E9743F8ED4F3139BB D0C94D1974975A4B9CD9EE6F18A21120 Nesticella brevipes (Yaginuma, 1970) Figs 5A-J , 7A-D , 16A (Japanese name: ko-horahimegumo コホラヒメグモ ) Theridion pilula Komatsu 1940 : 194, fig. 5a-d (♀, misidentification). Nesticus brevipes Yaginuma 1970 : 386, figs 1, 2 (♂); Yamaguchi and Yaginuma 1971 : 172, figs 1, 2 (♀); Yaginuma 1972 : 619, fig. 2 (♂♀); Irie 1981 : 31, figs 1-3 (♂♀); Chikuni 1989 : 45, fig. 3 (♂♀); Kamura and Irie 2009 : 353, fig. 106 (♀). Type locality. Japan Shikoku Is., Kochi Pref., Tosa-Yamada-cho, Sakagawa, Ryuga-dō cave (龍河洞). Material examined. Japan : Honshu Is.: Wakayama Pref. : 1♀ , Higashimuro-gun , Kushimoto , 23.Aug.1993 , A. Tanikawa leg. (FBPC); Shiga Pref. : 1♀ , Koga , Shigaraki-cho , Miyajiri , 28.Jun.2022 , M. Yoshida leg. (FBPC) ; 1♀ , Otsu , Sakamoto , 23.Jul.2022 , M. Yoshida leg. (FBPC) ; Shikoku Is.: Tokushima Pref : 2♀ , Tokushima , Nyūtachō , Konji , Konjiji temple (建治寺), narrow and dry tunnel in the cliff near the temple, 34.02769°N , 134.42923°E , 13.May.2019 , F. Ballarin leg. (FBPC); Kochi Pref. : 1♀ ( holotype ), Kami , Tosayamadacho Sakakawa , Ryuga-dō cave (龍河洞), 11.Apr.1970 , S. Ueno leg. (NMST-Ar.75) ; 1♂ ( paratype ), same data and locality (NMST-Ar.76) ; Kyushu Is.: Saga Pref. : 1♀ , Fujicho , 31.Jul.2005 , A. Tanikawa leg. (MNHAH); Kumamoto Pref. : 1♂ , 3♀ , Aso-gun , Aso-machi , Kikuchi Keikoku gorge, 8.Aug.2003 , T. Irie leg. (NSMT-Ar.5689); same locality, 11.Jul.2004 , T. Irie leg. (NSMT-Ar.5713) ; 1♂ , 2♀ , Kami-mashiki-gun, Tonochi-machi , Kashiwagawa , 23.May.2004 , T. Irie leg. (NMST-Ar.5728, identified as H. mogera ) ; 1♀ , Kuma-gun , Itsuki-mura , Otaki , 13.May.2004 , T. Irie leg. (NMST-Ar.5721); Ōita Pref. : 2♀ , Ōita-shi , Ochi Shimohetsugi , Ōzuru-doukustu-iseki cave (尾津留洞窟遺跡), 38 m large and rather dry cave, 33.16747°N , 131.67679°E , 17.Mar.2019 , F. Ballarin leg. (FBPC); Kagoshima Pref. : 2♀ , Minami Kyushu-shi , Kawabe-cho , 26.Feb.2007 , K. Ishii leg. (NMST-Ar.14585, identified as H. mogera ) ; 1♀ , Minamisatsuma-shi , Kasasa-cho ; 9.Dec.2007 , K. Ishii leg. (NSMT-Ar.14513) . Diagnosis. This species is closely related to N. terrestris and N. silvicola . Male of N. brevipes can be distinguished from male of the latter two species by the presence of two distal processes of paracymbium (Di-I-II), a sharper radical apophysis (Ra), and a thinner median process of conductor (Cm) (vs a single, sharper Di, a stockier Ra, and a thicker Cm in N. terrestris and N. silvicola ). (Figs 5A-D , 7A-C cf. Figs 6A-D , 7E-G , 8A-D , 10A-C ). In addition, the origin of the embolus (E) from the radix is located in a different position than in N. terrestris (4:30 o'clock in N. brevipes vs 6:00 o'clock in N. terrestris ) (Figs 5A , 7A cf. Figs 6A , 7E ). Figure 5. Nesticella brevipes A male palp (paratype), ventral view B same, retrolateral view C same, ventro-prolateral view D same, dorsal view E female epigyne (holotype), ventral view F same, female from Kyushu G vulva, dorsal view H habitus of male (old specimen from Kyushu); I habitus of female (specimen from Shikoku); J cephalic area of female, frontal view. Abbreviations: Cd - copulatory duct; Co - copulatory opening; Id - insemination duct; S - spermatheca; Sc - scapus. Scale bars: 0.2 mm ( A-G, J ); 1.0 mm ( H, I ). Figure 6. Nesticella terrestris A male palp (one of the topotypes), ventral view B same, retrolateral view C same, ventro-prolateral view D same, dorsal view E female epigyne (topotype), ventral view F same after dissection, shape variation G vulva, dorsal view H habitus of male (topotype) I habitus of female J cephalic area of female, frontal view. Abbreviations: Cd - copulatory duct; Co - copulatory opening; Id - insemination duct; S - spermatheca; Sc - scapus. Scale bars: 0.2 mm ( A-G, J ); 1.0 mm ( H, I ). Figure 7. Genitalia of Nesticella brevipes and N. terrestris A male palp of N. brevipes , ventral view B same, retrolateral view C detail or paracymbium, dorsal view E female epigyne, ventral view E male palp of N. terrestris , ventral view F same, retrolateral view C detail or paracymbium, dorsal view H female epigyne, ventral view. Abbreviations: Cl - lobe of conductor; Cm - median process of conductor; Co - copulatory opening; Cp - prolateral process of conductor; Cr - retrolateral process of conductor; Di I-II - distal process(es) I and II of paracymbium; Do - dorsal process of paracymbium; E - embolus; P - paracymbium; Ra - radical apophysis; Rx - radix; S - spermatheca; Sc - scapus; Sd - sperm duct; St - subtegulum; Te - tegulum; Ve I-II - ventral processes I and II of paracymbium. Scale bars: 0.2 mm. Figure 8. Nesticella silvicola sp. nov. A male palp (holotype), ventral view B same, retrolateral view C same, ventro-prolateral view D same, dorsal view E female epigyne (one of the paratypes), ventral view F same, from shape variation G vulva, dorsal view H habitus of male I habitus of female J cephalic area of female, frontal view. Abbreviations: Cd - copulatory duct; Co - copulatory opening; Id - insemination duct; S - spermatheca; Sc - scapus. Scale bars: 0.2 mm ( A-G, J ); 1.0 mm ( H, I ). Female of N. brevipes can be separated from female of N. terrestris and N. silvicola by the slimmer scapus (Sc), approximately as long as wide, usually with a more rounded posterior margin (vs a larger Sc, ~ 1.5-2.0 x wider than long, having a flatter posterior margin in N. terrestris and N. silvicola ) (Figs 5E, F , 7D cf. Figs 6E, F , 7H , 8E, F , 10D ). In addition, N. brevipes shows wider spermathecae (S) than the diameter of the copulatory ducts (Cd) (vs same diameter of Cd in N. terrestris and N. silvicola ) and internal ducts with a more convoluted and irregular trend (vs more straight ducts in N. terrestris or strongly bent in the middle in N. silvicola both with less clear coils). (Figs 5E-G cf. Figs 6E-G , 8E-G ). In addition, N. brevipes is generally smaller is size than N. terrestris (females 1.76-2.50 vs 2.31-3.30, see also Fig. 17A, B ). Redescription of male (paratype). Habitus as in Fig. 5H . Total length 1.94. Prosoma 1.02 long, 0.87 wide. Carapace rounded, uniformly brown-yellowish with borders and central area slightly darker. Cervical groove and fovea distinct. Chelicerae, labium, maxillae, and sternum of the same uniform color as carapace. Eyes well developed. Eyes measurements: AME = 0.03, ALE = 0.06, PME = 0.07, PLE = 0.07, AME-ALE = 0.04, ALE-PLE = 0.00. Legs uniformly pale yellowish. Legs measurements: I 7.28 (2.04, 0.47, 1.92, 2.04, 0.81), II 5.70 (1.70, 0.43, 1.44, 1.45, 0.68), III 4.23 (1.30, 0.38, 0.96, 1.00, 0.59), IV 5.56 (1.86, 0.43, 1.51, 1.18, 0.58). Opisthosoma greyish with large black marks on dorsal and frontal sides. Male palp as in Figs 5A-D , 7A-C . Cymbium relatively short, covered with sparse setae, several thicker setae on distal-prolateral margin (Fig. 5D ). Paracymbium with 2 hook-like distal processes (Di-I-II), 2 ventral processes (Ve-I-II), and a single dorsal apophysis (Do). Distal process I (Di-I) thick, headed antero-retrolaterally; distal process II (Di-II) slimmer and sharper, headed retrolaterally; ventral process I (Ve-I) short and stocky, headed internally; ventral process II (Ve-II) long and lobated, headed internally; dorsal apophysis (Do) lobated, wide and flat (Figs 5A-D , 7A-C ). Embolus (E) long and filiform, origin of embolus positioned at ~ 4:30 o'clock on radix (Rx). Radical apophysis (Ra) strongly sclerotized, triangular with a rather sharp tip. Conductor with 3 distinct processes (Cp, Cr, Cm) and a half-transparent distal lobe (Cl). Prolateral process of the conductor (Cp) flat, ribbon-like, headed counterclockwise and wrapped around embolus. Retrolateral process of conductor (Cr) wide and thick, curved inside. Median process of conductor (Cm) tiny and slim, strongly sclerotized with a ribbon-like lobe wrapped around its prolateral side (Figs 5A-C , 7A, B ). Redescription of female (holotype). Habitus (fresh specimen from Shikoku) as in Fig. 5I . Total length 2.24. Prosoma 1.02 long, 0.92 wide. Cephalic area as in Fig. 5J . Carapace piriform. Eyes measurements: AME = 0.02, ALE = 0.06, PME = 0.05, PLE = 0.06, AME-ALE = 0.05, ALE-PLE = 0.00. Coloration and other details as in male. Legs yellowish with darker annulation on femur and tibia. Legs measurements as follows: I 6.55 (1.88, 0.46, 1.74, 1.66, 0.81), II 5.07 (1.52, 0.40, 1.24, 1.21, 0.70), III 3.85 (1.20, 0.30, 0.85, 0.91, 0.59), IV 5.23 (1.67, 0.43, 1.31, 1.17, 0.65). Epigyne and vulva as in Figs 5E-G , 7D . Scapus (Sc) short and stumpy, approximately as long as wide, ending with a rounded, convex posterior margin (Figs 5E, F , 7D ). Copulatory opening (Co) at the inner-lateral sides of scapus. Internal ducts slightly visible through the transparent tegument, shaped as a narrow V. Copulatory ducts (Cd) short, straight, and thick, slightly divergent from each other. Insemination ducts thin, coiled around the copulatory ducts. Spermathecae (S) rounded, slightly wider than copulatory ducts, separated from each other by ~ 2 x their diameter (Fig. 5G ). Size variation. Male (based on 3 specimens): total length: 1.94-2.31, prosoma length: 1.02-1.17, prosoma width: 0.87-0.99. Female (based on 10 specimens): total length: 1.76-2.50, prosoma length: 0.90-1.15, prosoma width: 0.81-0.99. Distribution. Western Japan (Shikoku, Kyushu, western Honshu?), China? (Fig. 16A ). The easternmost limit of this species in Japan seems to be located in the Kansai area where it apparently overlaps with the distribution of N. terrestris (Fig. 16A ). The precise boundaries between these two species remain unclear. All samples and published drawings of N. brevipes from China, Korea, and Russian Far East checked by us refer to different species of Nesticella . In lack of clear records, the presence of this species outside Japan remains unconfirmed although it seems unlikely. See also "remarks on misidentifications" for additional information. Habitat and ecology. Nesticella brevipes can be found in humid and shadowed environments such as undergrowth of deciduous and evergreen forests growing in narrow valleys, vegetated cliffs, screes, caves, and artificial tunnels. This species builds simple scaffold webs in empty spaces among the leaf litter, between rocks or in the crevices in the walls and on the floor of caves. In subterranean environments we collected this species in both the twilight and the dark zones. Remarks on intraspecific variation. Like many other nesticid species N. brevipes shows a certain degree of intraspecific variation in the shape of genitalia and in particular in the shape of the scapus of the female epigyne. Some individuals from Shikoku Is. (e.g., from Kikuchi Keikoku Gorge), and especially populations from the Kansai area, show a more squared scapus with the posterior margin more flattened than normal. A high degree of intraspecific genetic divergence (7.6%) is also observed between the population living in Shikoku Is. and Kyushu Is. Remarks on misidentifications. Nesticella brevipes was first described and illustrated by Komatsu (1940) based on specimens from Ryuga-do cave in the island of Shikoku but wrongly identified as Theridion pilula (= Phoroncidia pilula (Karsch, 1879), Fam. Theridiidae Sundevall, 1833). Yaginuma (1970 : p. 386-388, figs 1, 2) recognized the species as new to science and described it based on specimens from the same cave. Nevertheless, he misidentified the male of the closely related N. terrestris thus reporting under N. brevipes a mix of specimens from the two species ( Yaginuma 1970 : p. 388, 390). Two years later Yaginuma (1972 : p. 619-621), in his revision of short-legged nesticids from Japan, synonymized N. terrestris with N. brevipes although it recognized them as belonging to different morpho-groups based on the morphology of the male palp and female epigyne. The outcome of our study, based on both morphological and molecular evidence, clearly supports the original separation of N. brevipes and N. terrestris as two distinct species. Since the time of its description Nesticella brevipes "sensu lato" has been frequently recorded by Japanese or foreign authors, in Japan and in other surrounding countries ( World Spider Catalog 2023 , see Shinkai et al. 2022 for the Japanese records). Nevertheless, due to the previous synonymization of N. terrestris with N. brevipes , it is difficult to understand to which species these records refer without directly checking the genitalia of the specimens. Thus, in this work we considered only the records of N. brevipes which samples have been directly examined by us or of which clear published drawings of genitalia were available. Based on the examined material we confirm the presence of N. brevipes in the island of Kyushu and Shikoku and in the Kansai area. We could not check any specimen from the Chugoku area thus the presence of this species in the western part of the Honshu Island, although possible, still needs to be properly confirmed. All records from central-eastern Honshu examined by us refer instead to the similar N. terrestris (see Fig. 16A ). Yaginuma (1970 , 1972 ) cited the presence of an unusual population of N. brevipes inhabiting some caves in Mie Prefecture (e.g., Fubonji-do cave). These specimens show partially reduced eyes ( Yaginuma 1970 : fig. 10) and, apparently, morphological characters of palp and epigyne mixed between those of N. brevipes and N. terrestris ( Yaginuma 1972 : p. 620, fig. 3). We did not have the opportunity to examine these specific specimens; however, other samples collected in both epigean and hypogean environments in Mie Pref. and checked by us refer to the similar N. terrestris . It is possible that the Nesticella from Fubonji-do cave represent a different and still undescribed troglobitic species. Irie (1981) , in his work on cave spiders from Kyushu, illustrate a population of N. brevipes from the Kikuchi Keikoku Gorge, northern Kumamoto Pref., which female show a wide and squared scapus ( Irie 1981 , figs 2, 3). These specimens were examined by us and, although some individuals have the scapus more squared and sometimes wider than usual, both males and female show the diagnostic characters of N. brevipes . Concerning the records outside Japan, specimens of N. brevipes from Kuril Is. ( Marusik and Crawford 2006 ) were inspected by us and they refer to N. terrestris . We did not have the possibility to check samples from Korea, nevertheless none of the published drawings of Korean specimens show the diagnostic characters of N. brevipes . Based on the shape of the epigyne they clearly refer to H. mogera (cf. Figs 1A-G , 4A-D vs Paik 1996 : figs 6, 9, 10) or to other species of Nesticella of the N. brevipes group, including possibly N. terrestris (cf. Figs 5A-G , 7A-D vs Figs 6A-G , 7E-H vs Paik 1996 : figs 7, 8 vs Namkung 2002 : fig. 80a, b vs Kim and Lee 2018 : fig. 27b, c). On the basis of these observations, we consider the presence of N. brevipes in Korea unlikely. During his previous studies, the first author had the occasion to examine several Nesticella specimens from China identified as N. brevipes and preserved in the collections of the Chinese Academy of Sciences, Beijing. All these specimens were revealed to be misidentifications of other endemic Nesticella or Howaia species. For example, the specimens determined as N. brevipes by Zhang and Li (2013) were recognized by the first author as H. huomachongensis (Lin, Ballarin & Li, 2016). The drawing of the male palp of N. brevipes from Zhejiang, China, published in Song et al. (1999) do not allow a clear identification of the species, it may refer to N. brevipes as well as another similar Chinese species of the N. brevipes group which was not yet described at the time of the publication of the book. Considering such circumstances, the presence of N. brevipes in China is unlikely but needs to be confirmed.